Mass spectrometric determination of dioxygen bond splitting in the "peroxy" intermediate of cytochrome c oxidase

Abstract

The "peroxy" intermediate (P form) of bovine cytochrome c oxidase was prepared by reaction of the two-electron reduced mixed-valence CO complex with (18)O(2) after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for (18)O enrichment by mass spectrometry. It was found that approximately one oxygen atom ((18)O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen-oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water.

Figure 1

Dependence of sample enrichment [δ¹⁸O] on ¹⁸O concentration for H₂¹⁸O (●); the solid line is a linear regression on these points. The average enrichment for the P samples vs. the concentration of P is plotted on the same graph (■). The interpolation of the mass spectrometric signal of P form onto the straight line indicates that the average H₂¹⁸O content of three P samples was 210 μM; the average concentration of P determined optically was 228 ± 7 μM.

Scheme 1

Transition of two-electron reduced mixed-valence CO complex of CcO to the “peroxy” intermediate.